Development of sandwich-form biosensor to detect Mycobacterium tuberculosis complex in clinical sputum specimens

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The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Original

article

Development

of

sandwich-form

biosensor

to

detect

Mycobacterium

tuberculosis

complex

in

clinical

sputum

specimens

Taha

Roodbar

Shojaei

a

_,

_Mohamad

_Amran

_Mohd

_Salleh

a,∗

_,

_Meisam

_Tabatabaei

b

_,

Alireza

Ekrami

c

_,

_Roya

_Motallebi

d

_,

_Tavoos

_{Rahmani-Cherati}

e

_,

_Abdollah

_Hajalilou

a

_,

Raheleh

Jorﬁ

a

a_Institute_of_Advanced_Technology_(ITMA),_Universiti_Putra_Malaysia,₄₃₄₀₀_Serdang,_Selangor,_Malaysia

b_Nanosystems_Research_Team_(NRTeam),_Microbial_{Biotechnology}_and_Biosafety_Department,_Agricultural_{Biotechnology}_Research

InstituteofIran(ABRII),Karaj,Iran

c_Infectious_and_Tropical_Diseases_Research_Center,_Ahvaz_Jundishapur_University_of_Medical_Sciences,_Ahvaz,_Iran

d_Department_of_Plant_Breeding_and_{Biotechnology,}_College_of_Agriculture,_Shahrekord_University,_P.O._Box_115,_Shahrekord,_Iran e_Research_and_Development_Department,_Nanozino,_16536-43181_Tehran,_Iran

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30January2014 Accepted19May2014

Availableonline30August2014

Keywords:

Mycobacteriumtuberculosiscomplex (MTBC)

Cadmium-telluridequantumdots (CdTe-QDs)

Goldnanoparticles(AuNPs) Fluorescenceresonanceenergy transfer(FRET)

Sandwich-formFRET-based biosensor

a

b

s

t

r

a

c

t

Mycobacteriumtuberculosis,thecausingagentoftuberculosis,comessecondonlyafterHIV onthelistofinfectiousagentsslaughteringmanyworldwide.Duetothelimitationsbehind theconventionaldetectionmethods,itisthereforecriticaltodevelopnewsensitivesensing systemscapableofquickdetectionoftheinfectiousagent.Inthepresentstudy,the sur-facemodifiedcadmium-telluridequantumdotsandgoldnanoparticlesconjunctwithtwo specificoligonucleotidesagainstearlysecretoryantigenictarget6wereusedtodevelop a sandwich-form fluorescence resonanceenergy transfer-based biosensor to detect M. tuberculosiscomplexanddifferentiateM.tuberculosisandM.bovisBacilleCalmette–Guerin simultaneously.Thesensitivityandspecificityofthenewlydevelopedbiosensorwere94.2% and86.6%,respectively,whilethesensitivityandspecificityofpolymerasechainreaction andnestedpolymerasechainreactionwereconsiderablylower,74.2%,73.3%and82.8%, 80%,respectively.Thedetectionlimitsofthesandwich-formfluorescenceresonanceenergy transfer-basedbiosensorwerefarlower(10fg)thanthoseofthepolymerasechain reac-tionandnestedpolymerasechainreaction(100fg).Although thecostofthedeveloped nanobiosensorwasslightlyhigherthanthoseofthepolymerasechainreaction-based tech-niques,itsuniqueadvantagesintermsofturnaroundtime,highersensitivityandspecificity, aswellasa10-foldlowerdetectionlimitwouldclearlyrecommendthistestasamore appropriateandcost-effectivetoolforlargescaleoperations.

∗ _{Corresponding}_author.

E-mailaddresses:asalleh@upm.edu.my,amransalleh@yahoo.co.uk(M.A.MohdSalleh). http://dx.doi.org/10.1016/j.bjid.2014.05.015

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Introduction

Tuberculosis (TB) is one of the major chronic infectious diseasescausedbyMycobacteriumtuberculosis(MTB)and rep-resentsaglobalhealthconcern.1,2_It_is_posing_a_threat_even_in thedevelopedcountries,becauseitmayemergeasan obsta-cleofhumanimmunedeﬁciencysyndrome.2_TB_is_spread_in the airand canaffect all partsofhumanbody but mostly lungs.3_MTB_was_reported_to_have_caused_1.4_million_deaths and8.5millionincident casesacross theworldonlyinthe year2011.3

Beside MTB, the other members of what is commonly referred to as the M. tuberculosis complex including M. africanum,M.microti,andM.bovismayalsocauseTBinfection. TheonlyavailablevaccineagainstMTBistheattenuatedM. bovisstrainsorBacilleCalmette–Guerin(BCG)whichcauses some cross-reactivity or false-positive results during the detectionprocess.1,4 _Therefore, _developing_an_accurate_and reliabledetectiontechniquecapableofdifferentiatinginfected samplesfromthosevaccinatedisalsorequired.

Ontheotherhand,earlydetectionissocriticaltoavoid aTBepidemic.Toachievethat,manytechniqueshavebeen developedandwidelyappliedtodatesuchasbacteria’s phys-ical,physiologicalandbiochemicalcharacteristicsaswellas polymerasechainreaction(PCR)-basedtechniques.2,5–11_More recentlyWHOrecommendedXpertMTB/RIFdetection tech-niqueasaprimarysensitivediagnostic test.12 _However,_all thesetechniqueshaveanumber ofshortcomings andas a result,thereisstillagrowingdesiretoaccomplishasimple, rapid,sensitiveand speciﬁcdetectionmethodto differenti-ateMTBC-infectedsamplesfromvaccinatedsampleswithan affordablecost.2,8,11,13,14

Theconserved genomic region of 6-kDaearly secretory antigenictarget 6(ESAT-6) has been foundof high homol-ogy among the different species ofmycobacteria and has beenused inthemost studiestodetect MTBC.15,16 _On_the other hand, to differentiate BCG vaccinated samples from MTB-infected samples, the ESAT-6 genomic region which is eliminated in all available BCG strains but present in the MTBcomplex was utilized.15,16 _In_addition, _during_the last decade, by applying nano-sized materials in biologi-cal detection and biological imaging aspects, the clinical diagnostics field has improved dramatically by developing rapid and sensitive methods at lower cost.17,18 Semicon-ductor nanocrystals, also known as quantum dots (QDs), are one of the most attractive fluorescent nanoparticles whichhavebeenwidelyappliedinbio-detectionprocesses.19 QDs are considered as alternative fluorescent probes for their unique optical properties such as high fluorescence yields, high photo-stability and narrow symmetric emis-sionspectrum.19 _Moreover,_the_symmetric_emission_spectra of QDs have nominated them as an appropriate donor moleculeforFörsterresonanceenergytransfer(FRET)-based sensors,18,20,21_in_which_the_electronic_excitation_energy_of_a donormoleculeistransferredtoanearbyacceptormolecule via adipole–dipoleinteraction betweenthe donor/acceptor pair.22_Case_in_point,_the_QDs_broadly_used_as_bio-sensors_by immobilizingwithparticularprobestodetectaspecifictarget nucleotide.23

Goldnanoparticles(AuNPs)havealsobeenutilizedwidely in the detection ofspecific RNA or DNA moleculesdue to theirunique opticalproperties.24,25 _AuNPs_have_remarkably highextinctioncoefficientsandabroadabsorptionspectrum, allowing higher sensitivity in optical detection techniques than traditionaldyes and designating AuNPsas a suitable acceptorforFRET.23,26_Due_to_the_fact_that,_the_emission_and absorption spectrum of CdTe-QDs and AuNPs significantly overlapin530nm,theemissionoftheQDsisquenchedwhen associatedwithoppositelychargedAuNPs.23,27

Inthepresentstudy,QDswereimmobilizedwithaspecific oligonucleotide (P1) andAuNPs wasconjunctwith another specificoligonucleotide(P2)againstESAT-6regiontodevelop aspecificandsensitivesandwich-formFRET-basedbiosensor todetectanddifferentiateM.tuberculosiscomplexfromBCG rapidly, accurately,and economically(Fig.1). Moreover,the validityofsandwich-formFRET-basedbiosensorin compar-isonwithculture,PCRandNestedPCRwasevaluated.

Materials

and

methods

Samples

Inthepresentstudy,50clinicalsamples(allsubjectswereHIV negative)werecollectedfromsputumspecimensofpatients whoweresuspectedtohavetuberculosisinTehranprovince hospitals, Iran (betweenDecember of 2005 and November 2008). The used procedures in the present study were in accordancewiththeethicalstandardsoftheresponsible com-mittee on humanexperimentation from each participating hospital.Thepatientshavegiveninformed priorto partici-patingintheresearch.Thedecontaminationandcultivation ofsampleswerecarriedout inthehospitals.Sampleswere decontaminated byusingN-acetylcysteine–NaOHprocedures. The mentioned samples were earlier analyzed and deter-mined byusing acultivation techniqueasa gold-standard detection methodby incubation250␮LofMycobacterium in theLowenstein–Jensenmediaat37◦Cinhumidatmosphere containing5%CO2.28

Primersandprobes

In the PCR-based-detection phases, the primer sets were orderedbased onaninsertionsequence oftheIS6110gene region(Table1).29,30

Inthenano-baseddetectionphase,twodifferent oligonu-cleotideprobeswere usedbasedontheconservedgenomic regionsofESAT-616_to_detect_MTBC_and_{differentiate}_between BCGandotherMycobacteriumspeciessimultaneously(Table1). The5endoftheﬁrstoligonucleotideprobe(P1)waslinkedto QDsbyNH2andthe3endofthesecondoligonucleotideprobe

(P2) waslinked toAuNPsbySH tofacilitatethe hybridiza-tion process. The probes were purchased from Invitrogen (ShanghaiInvitrogenBiotechniqueCo).

DNAdetectionbyPCRandnestedPCRassays

According to a DNA extraction technique described elsewhere,31 _the_chromosomal_DNA _was_extracted_utilizing

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QD QD QD QD QD Au Au Au Au Au FRET Probe 1 Probe 2 Complementary target DNA Uncomplementary DNA

Fig.1–Sandwich-formFRET-basedbiosensorschematic.InthepresenceofthetargettheAuNPs/P2moietyoptically quenchedtheQDs/P1moiety.PresenceofthetargetmoleculesturnedtheAuNPs/P2intoaﬂuorescenceacceptorclose enoughtotheQDs/P1resultinginaFRETsignal.

Table1–CharacteristicsofDNAoligonucleotideprimers andprobesutilizedfordetectionofMTBCbyPCR,nested PCRandnanobiosensor.

Technique Sequence(5→3) Genomic

region

PCR R:CCTGCGAGCGTAGGCGTCGG

F:CTCGTCCAGCGCCGCTTCGG

IS6110 NestedPCR Outerset

Pt8:GTGCGGATGGTCGCAGAGAT Pt9:GTGCGGATGGTCGCAGAGAT Innerset TB290: GGCGGGACAACGCCGAAT-GCGAA Pt9:GTGCGGATGGTCGCAGAGAT IS6110 Pre-preparation R: ACGAAGCTTTGCGAACATCCCAGT-GACGTT F: AATCGGATCCATGACA-GAGCAGCAGTGGAATTTC ESAT-6 ESAT-6 Nanobiosensor R: ACGAAGCTTTGCGAACATCCCAGT-GACGTT F: AATCGGATCCATGACA-GAGCAGCAGTGGAATTTC P1:NH2 (CH2)6-GTAAGTAAGGGAGGAAC P2:TGCTCCCCTTCGTCAGG-(CH2)6-SH ESAT-6 ESAT-6 ESAT-6 ESAT-6

proteinaseKand phenol:chloroform,and wassubsequently precipitated by using isopropanol and ethanol. To detect MTBCDNA,thePCRandnestedPCRassayswereperformed by utilizing BioRad thermocycler (BioRad, CA, USA) based on theprotocols described byEisenachet al.32 _and _Wilson et al.33 _To_examine_the_existence_and_size_of_the_ampliﬁed fragments,2%agarosegelandtheGeneRulerTM 50bpDNA Ladder (Fermentas)were used. Visualizationofagarose gel wasdonebyethidiumbromideandgelswerephotographed withUVPBioDoc-ItTMsystemCCDcamera(UVPInc.,Upland, USA).Moreover,todeterminethedetectionlimitsoftheused methods,differentdilutionsofaknownchromosomalDNA stockwereused.

SynthesisofAuNPs

TheAuNPs weresynthesized through thecitratereduction method by adding trisodium citratesolution to 170mgL−1 HAuC14 under stirring and temperature condition. After a

color shift insolution from yellow to red, the stirring and heatingwere stoppedandthesolutionwasstoredat4◦C.24 Afterwards, the synthesized AuNPs were characterized by transmissionelectronmicroscope(TEM)(HitachiH-7100)and particles diameter size were determined using UTHSCSA ImageTool(UniversityofTexas).

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SynthesisofCdTeQDs

TheNaHTesolutionwaspreparedbyadding2mMofNaBH4 andtelluriumpowderin50mLofdistilledwater.Toprepare CdTeQDs,10mMofCdC12and25mMofTGAweredissolvedin

250mLdistilledwaterunderstirringandthepHwasadjusted to11byusingNaOHsolution.Subsequently,theNaHTe solu-tion was addedto the solution underN2 protectionunder

stirringfor25min.Finally,thismixturewasheatedtoboilat 100◦Cfor2h.34 _Then,_the _synthesized_AuNPs_were charac-terizedbyTEM(HitachiH-7100)andparticles diametersize weredetermined usingUTHSCSAImageTool(Universityof Texas).

Conjugationoftheﬁrstoligonucleotide(P1)ontheCdTe QDssurface

Conjugationofthe ﬁrst probe on the surface ofCdTe QDs was carried out by mixing 1mL of CdTe QDs, 20␮g of the P1 and 50␮L of 1-ethyl-3-(dimethylaminopropyl) car-bodiimide hydrochloride(EDC). Thesolution was mixed in 50mMdipotassiumphosphatebuffersolution(pH=6)atroom temperature.23

Assemblingofthesecondoligonucleotide(P2)onthe AuNPssurface

Topreparetheself-assemblyoftheprobes ontothe AuNPs surface,10mLofAuNPssolution and 20␮gofthe P2were mixedatroomtemperatureforabout 10h.Afterwards, the solution was added into a dipotassium phosphate buffer solution(pH=6.0).TopurifytheAuNPs-probe,centrifugation wasperformedat18,000rcffor25minandthepelletwas re-suspendedindipotassiumphosphatebuffersolution.23

Pre-preparationofclinicalsputumspecimensforbiosensor procedure

TheclinicalsputumsampleswereﬁrstusedforPCR ampliﬁ-cationofESAT-6gene(Table1).

ThePCRwasperformedbyutilizingBioRadthermocycler (BioRad,CA,USA)basedontheprotocolsdescribedby Eise-nachet al.32 _{Subsequently,}_PCR_products_were _analyzed_by sandwich-formFRET-basedbiosensor.

Sandwich-formFRET-basedbiosensor

Toperformthe sandwich-formFRET-basedbiosensorassay, 10␮L of QDs/P1 solution, 10␮L of AuNPs/P2 solution and 10␮L ofPCR product were mixed in atube. Subsequently, denaturationandannealingofsampleswiththeimmobilized oligonucleotideswere carried out at94◦C fortwominand at65–68◦C for twomin, respectively, using aBioRad ther-mocycler(BioRad,CA,USA).Then,theﬂuorescenceintention spectrawererecordedbyusingaspectrophotometer.Baseline changesestimatedbysubtractingthesecondrunfromtheﬁrst runandthethresholdfornegativesamplewasestimatedat 8.2±3.

Fluorescenceprocedure

The ﬂuorescence spectra were monitored by using a PD-3000UVultraviolet–visiblespectrophotometer(Appel,Japan). Theexcitationwavelength(excitationwavelengthofQDs)was setat370nmandtheemissionspectrawereprobedbetween 400and680nmastheAuNP’s(quencher)emissionwaslocated intherangeof570–650nm.Theexcitationandemission band-widthof5nmwasused.

Dataanalysis

DataanalysiswasperformedusingtheSAS6.12(SAS Insti-tute,Cary,NC),anddiagnosticsensitivityandspeciﬁcitywere determinedusingusualformula.

Sensitivity= TruePositive

TruePositive+FalseNegative×100 Speciﬁcity=_False_PositiveTrueNegative₊_True_Negative×100

Theotherstatisticalparameterswerecalculatedusingan onlineclinicalcalculatoravailableathttp://www.vassarstats. net/clin1.html.

Results

Asmentionedearlier,cultivationtechniquewasconsidered thegold-standarddetectionmethodinordertocomparethe othertechniquesused,i.e.PCR,nestedPCRandthe sandwich-form FRET-based biosensor. In presence of M. tuberculosis infection in the sample, PCR and nested PCR products of about123bpand360bpwere observedusingIS6110primer sets (Fig. 2), and 60% (30/50)and 64% (32/50) ofthe speci-menswerefoundpositive,respectively.On theotherhand, outofthe50samplestestedbythesandwich-formFRET-based biosensor,70%(35/50)werefoundpositive.Interestingly,out of those 35 positive samples bythe FRET-based biosensor, only33(94.2%)wereculture-positive.Thus,twopositive sam-plesbyFRET-basedbiosensorassaywerereportednegativeby theculture-basedmethod.Furthermore,outofthe35positive samples,only27(77.1%)and30(85.7%)weredetectedbyPCR andnestedPCRtechniques,respectively.

Therefore,theoverallsensitivityandspecificityofthePCR method inrelationtothe cultivationmethod fordetection of MTBC was 74.2% (95% CI 56.4–86.8) and 73.3% (95% CI 44.8–91.0),respectively(Table2).Besides,sensitivityand speci-ficityofthenestedPCRmethodinrelationtothecultivation technique were 82.8% (95% CI 65.7–92.8) and 80% (95% CI 51.3–94.6),respectively(Table2).Thesampleswerefirst uti-lizedforPCRamplificationofESAT-6geneandsubsequently PCR productswere analyzedbysandwich-formFRET-based biosensor.Theresultsobtainedrevealedthatthe sandwich-formFRET-basedbiosensorversuscultivationdemonstrated indetectingMycobacteriumhad sensitivityandspecificityof 94.2%(95%CI79.4–99.0)and86.6%(95%CI58.3–97.6), respec-tively(Table2).

Todeterminethecharacteristicsofthedonorsand accep-tors in the sandwich-form FRET-based biosensor, the QDs andAuNPswerecharacterizedbyusingTEM.TheTEMimage

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A

B

360

123

M

1

2

3

4

3

2

1 M

Fig.2–AmpliﬁedproductsofMTBCbyPCRandnestedPCRmethods.(A)PCRproductsM:GeneRulerTM₅₀_bp_DNA_Ladder,

1:positivecontrolsample,2:clinicalpositivesample,3:negativecontrolsample,4:clinicalnegativesample.(B)NestedPCR products(secondround)M:GeneRulerTM₅₀_bp_DNA_Ladder,_1:_clinical_positive_sample,_2:_positive_control_sample,_3:

negativecontrolsample,4:clinicalnegativesample.

50 nm

A

_B

50 nm

Fig.3–TEMimageofsynthesizedAuNPs(A)andCdTe-QDs(B).

showedthattheQDand Aunanoparticleshaveaspherical morphologyandsuitablemonodispersity(Fig.3AandB).The QDsand AuNPsparticlesizewere determined at6nmand 8nm,respectively.Fromthemechanisticpointofview,inthe

solutioncontainingonlytheQDs/P1andAuNPs/P2(negative control), the ﬂuorescencespectrum wasrecorded at50AU, whileinpresenceofMycobacteriumDNA(targetmolecule),a signiﬁcantdownwardshiftinthespectrumpeakingat10AU

Table2–ComparisonbetweenFRET-basedbiosensor,PCRandnestedPCRversusculturemethodfordetectionof Mycobacteriumtuberculosisin50sputumclinicalsamples.

Methods Cultivationassay Value

Positive(n) Negative(n) Sensitivity% Speciﬁcity%

FRET-basednanobiosensor Positive 33 2 94.2 86.6 Negative 2 13 Total 35 15 PCR Positive 26 4 74.2 73.3 Negative 9 11 Total 35 15 NestedPCR Positive 29 3 82.8 80.0 Negative 6 12 Total 35 15

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Wavelength (nm) Fluorescence intensity (A U) 520 50 10 590 680

QDs/P1 + AuNPs/P2 + Positive sample QDs/P1 + AuNPs/P2 or QDs/P1 + AuNPs/P2 + Negative sample

Fig.4–FluorometricpeaksofQDs/P1+AuNPs/P2solution andsolutioncontainedQDs/P1+AuNPs/P2+positive sampleobtainedbyspectrophotometer.

wasobserved(Fig.4).TheemissionspectrumoftheQDsand absorptionspectrumofAuNPsdemonstratedmaximum over-lap thatis critical toobtain FRET phenomena (Fig. 5). The FRETefficiency(E) ofthepresentedsystemwasdependent onmolarratioofAuNPs/P2toQDs/P1.Increasesinthemolar ratioofAuNPs/P2toQDs/P1leadstoanincreaseintheFRET efficiencyandthehighestFRETefficiencyfordeveloped sys-temwasachievedatAuNPs/P2toQDs/P1molarrationof1:10 (Fig.6) 400 0 0,2 0,4 0,6 0,8 1 450 500 550 Wavelength (nm) Nor maliz

ed absorbance intensity (a.u.)

QDs Em. AuNPs Abs.

600 650

Fig.5–OverlapbetweenQDsemissionspectrumand AuNPsabsorptionspectrum.

QDs-Prob1/AuNPs-Prob2 ratio 80 60 40 20 0:0 1:1 1:5 1:10 1:15 1:20 FRET efficiency %

Fig.6–Energytransferefﬁciencyfromdonor(QDs)toan acceptor(AuNPs)indifferentmolarratioofAuNPs/P2to QDs/P1.

Table3–ComparisonbetweenPCR,RT-PCRand

nanobiosensordetectionlimits.Presenceandabsenceof speciﬁcbandinagarosegelinPCRandRT-PCRdetection methodsweresignedpositive(+)andnegative(−) respectively.Inthenanobiosensortechnique,

observationofdownwardshiftintheﬂuorometercurve wassignedaspositive(presenceoftarget)andnoshift intheﬂuorometercurvewassignedasnegative (absenceoftarget). Sample Volume (fgmL−1) PCR RTPCR Nanobiosensor Positivecontrol 200 + + + 150 + + + 100 + + + 50 − − + 30 − − + 20 − − + 10 − − + 5 − − − Negativecontrol 0 − − −

Finally, the detection limit of the PCR and nested PCR methodswasmeasuredat100fgwhileforthesandwich-form FRET-basedbiosensoritwas10-foldlowerat10fg(Table3).

Discussion

In theoligonucleotide-based detection systems, the impor-tant matter concerned is probes speciﬁcity against closely relatedspecies,andinthecaseofMTBCdetection,the dif-ferentiation betweenBCG vaccinated samplesand infected samples.Therefore,utilizingoligonucleotideprobesbasedon conservedregionofMTBC,whichisdeleted inthe M.bovis BCG,isimportant.Thispointwastakenintocareful consider-ationinthepresentstudy.Therefore,theconservedgenomic regionsofESAT-6wasusedastheprobetodetectMTBCand

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differentiateM.bovisBCGfromotherMycobacteriumspecies, duetothehigh existinghomologyintheESAT-6regionsof differentMycobacterialspeciesandtheabsenceofthisregion inBCGstrains.

Although,recentlyWHOrecommendedXpertMTB/RIFasa sensitivedetectionmethod,itisnotavailableinallcountries. Moreover,thewidelyusedmethodsinthecaseofclinicalTB detection inthe region inwhich the study was conducted arecultivationtestandnestedPCR;therefore,inthisregion thenewlydevelopedtechniquesmostlycomparedwiththese methods.Duetotheinsufficientsensitivityandspecificityof thePCR-basedassays,developingamoresensitivemethodfor thedetectionofMTBChasbeenconstantlysought.Although, thereare different valuesreported inthe published litera-ture, the sensitivity and specificity ofPCR assays used for detectionofMycobacteriuminthepresentstudywerein agree-mentwiththefindingsofChawlaetal.35_and_Ekrami_et_al.28 In the used clinical samples, four cases were found posi-tivebyculturebutnegativebyPCRand nested-PCR,which couldbeconsideredasfalsenegativeresults.Theseobserved falseresultswere attributedtothe concentration oftarget moleculesintheclinicalsamplesandtothespecificityof uti-lizedprimers.

In the case of the nested PCR assay, the sensitivity of 82.8%showedthatthis techniquecould notberegarded as anidealtechniquetodetectMTBC,despiteofitsspeciﬁcity tobeapproximatelyequaltothatofthesandwich-form FRET-basedbiosensor.ThelowestdetectionlimitofMycobacterium DNAachievedbyusingPCRandnestedPCRtechniqueswas 100fg,whichwasinagreementwithearlierreports.36,37_This valuewasremarkablyhigherthanthatachievedbyusingthe sandwich-formFRET-basedbiosensor(10fg).

In the sandwich-form FRET-based biosensor, the NH2

linkedoligonucleotide(P1)wascapableofcovalentlyattaching ontothesurface-modiﬁedQDswithEDCduetothe adsorp-tion tendency of the carboxyl group of EDC and the NH2

linked to the P1.38 _This _conjugate _showed _maximum flu-orescence radiation at548nm. Secondoligonucleotide (P2) could easily self-assemble on AuNPs due to the attraction ofSH linked tothe P2and surface ofAuNPs.39 _Two sam-plesthatwerepositivebycultureandnegativebynanosensor showed autofluorescent signals during examination. This problemhasbeenpreviouslyreportedbyEkramietal.28 Prob-ably, thereisa specificproblemwhen clinical samplesare tested.Generally,observedinterfacebyautofluorescencecan beattributedtonaturalfactorsinducedbysomereagentsin clinicalsamples.28

TheTEMimageconfirmedthat thesynthesized QDand Au nanoparticles were appropriate for the nanobiosensor designed (Fig. 3). The emission and absorption spectra of CdTe-QDsandAuNPssignificantlyoverlapat530nm; there-fore,CdTe-QDs and AuNPscould beusedsuccessfullyas a suitabledonorandacceptorpair(Fig.5).Tofindoutthe opti-mumFRET signal and maximum FRET efficiency, different molarratiosofAuNPs/P2toQDs/P1wereinvestigated. Opti-mummolarratioofAuNPs/P2toQDs/P1wasachievedat1:10 inwhich FRET efficiency wasabout 60% (Fig. 6). Athigher molarratios,manyoligonucleotideprobesimmobilizedinthe surfaceofAuNPswouldremainunhybridizedandtheFRET efficiencydecreasedenormously.While,atthelower molar

ratio,decreaseintheamountsofAuNPsleadstoinsufficient energytransferfromQDsnearAuNPsmoleculesand subse-quentlycausedareductionintheFRETsignal(Fig.6).More specifically,thefluorescencespectrumwas50AUforthe solu-tioncontainingQDs/P1andAuNPs/P2,butinthepresenceof thetargetadownwardshiftto10AUintheemissionspectrum wasrecorded (Fig.4).Theobserveddecreaseinthe fluores-cenceintensityinthesolutioncontainingthetargetsindicated that FRET process occurred in the QDs/P1-target-AuNPs/P2 complexsystem.Infact,theAuNPs/P2moietyinthepresence ofthetarget,opticallyquenchedtheQDs/P1moiety,basedon theForsterdipole–dipoleinteractionform.27,40_In_other_words, presenceofthetargetmoleculesturnedtheAuNPs/P2intoa fluorescenceacceptorcloseenoughtotheQDs/P1resultingin afivefoldFRETsignal(Fig.1).Thisprincipleledtoa down-ward shiftofthefluorometercurve incomparisonwiththe curveoriginallyobtainedforthesolutionwithoutanytarget DNA.

To the best ofour knowledge, there has been no pub-lishedreportsoncombiningQDsandAuNPstodetectbacterial targets, although some researchers have developed tech-niquesbyusingQDsandAuNPsseparately.41–43_For_instance, Soo etal.42 introducedanAuNPscross-linkingapproachto detectMTBC.Morespecifically,theAuNPswerefunctionalized withthiolmodifiedspecificprobes.Inthepresenceoftarget DNA,AuNPshybridizedtothetargetresultinginaggregation anddecreaseinabsorbanceofthesolution.Theabsorbance decreaseswereaccompaniedbyashiftinsolutioncolorfrom redtoreddishpurple.Ontheotherhand,intheabsenceof targetDNA,thecolorandabsorbanceofthesolutionwerenot altered.Thisassaydemonstratedthedetectionlimitofaslow as0.5pmoloftarget.42

Inanother study,Gazoulietal.43 _developed_a_new tech-niquecombining QDs withmagneticbeads(MBs) todetect M.tuberculosisand M.aviumsubsp.Paratuberculosisbyusing DNA extracted from bronchoalveolar lavage specimens. In thistechnique,twobiotinylatedoligonucleotideprobeswere immobilizedontheQDsandMBssurfacetoformasandwich likehybridization system.Intheabsenceoftarget,no ﬂuo-rescencesignalwasmonitoredwhileinthepresenceoftarget DNA,redﬂuorescencecolorwasobserved.Thedetectionlimit achievedinthisassaywasreportedat12.5ng.43

The developed sandwich-form FRET-based biosensor requires small amountofsample;therefore, itcould bean appropriate detection technique where limited volumesof samplesareavailable.Moreover,thenewlydeveloped biosen-sorincomparisonwiththecultivationmethodshowedhigh sensitivity andspeciﬁcity. Inaddition, the developed FRET-basedbiosensorpresentedadetectionlimitof10fgwhichwas remarkablylowerthanthe100fgofPCRandnestedPCR.The costofsandwich-formFRET-basedbiosensordevelopedinthe presentstudywasestimatedtobeslightlymorethanthe PCR-basedtechniques;butduetothespeedandaccuracyofthe FRET-basednanobiosensor,itslarge-scaleapplicationwould bemorecost-effective.

Conﬂicts

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